Biomarkers for risk prediction of mortality

ABSTRACT

The present invention relates to a method for predicting the risk of a subject of rapidly progressing to chronic heart failure and/or of hospitalization due to chronic heart failure and/or death. The method is based on the determination of at least one biomarker selected from the group consisting of a BNP-type peptide, IGFBP 7  (IGF binding protein  7 ), a cardiac Troponin, soluble ST 2  (sST 2 ), FGF- 23  (Fibroblast Growth Factor  23 ), and Growth Differentiation Factor  15  (GDF- 15 ), in a sample of a subject. The method may further encompass the assessment of the presence or absence of (i) abnormal midwall fractional shortening or (ii) left ventricular hypertrophy. Further envisaged by the present invention are devices adapted to carry out the present invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/581,587 filed Apr. 28, 2017, which is a continuation of InternationalApplication No. PCT/EP2015/075012 filed Oct. 28, 2015, which claimspriority to European Application Nos. 14192653.5 filed Nov. 11, 2014,and 14190836.8 filed Oct. 29, 2014, the disclosures of which are herebyincorporated by reference in their entirety.

BACKGROUND

An aim of modern medicine is to provide personalized or individualizedtreatment regimens. Those are treatment regimens which take into accounta patient's individual needs or risks. Personalized or individualtreatment regimens shall be even taken into account for measures whereit is required to decide on potential treatment regimens.

Heart failure (HF) is a major and growing public health problem. It isestimated that approximately 5 million patients in the USA have HF, morethan 500 000 patients are diagnosed with HF for the first time eachyear, and more than 250.000 patients in the US die each year of HF as aprimary cause. Heart failure (HF) is one of the main causes of morbidityand mortality in developed countries. Because of aging of the populationand greater longevity of patients with cardiovascular disease incidenceand prevalence of HF are increasing.

Heart failure may be symptomatic or asymptomatic. It is known somesubjects with asymptomatic heart failure progress more rapidly tochronic heart failure, and thus are at elevated risk of hospitalizationdue to heart failure and/or death (Neeland et al., Journal of theAmerican College of Cardiology Vol. 61, No. 2, 2013). It is important toidentify these subjects as early as possible since this would allow fortherapeutic measures that prevent or delay the progression to chronicheart failure. The identification of rapid disease progressors andappropriate therapeutic intervention is a, however, major unmet medicalneed (Neeland et al.).

Neeland et al. determines of cardiac markers in patients with leftventricular hypertrophy. Based on the determination of cardiac markers,a high-risk group in the general population can be identified.

WO 2014/040759 discloses that cardiac markers such as NT-proBNP and acardiac Troponin can used for the identification of subjects who shouldbe subjected to an imaging based diagnostic assessment.

Zapolski et al. describe the association of abnormal MFS (Midwallfractinonal shortening) and elevated levels of NTproBNP in across-sectional study performed in hemodialyzed patients. Theinvestigated patients are severely ill (Zapolski et al., BMC CardiovascDisord. 2012, 12:100).

Satyan et al. provide data of elevated NTproBNP in combination withabnormal MFS for the identification of the mortality risk inhemodialysis patients (Satyan S, Light R P, Agarwal R Am J Kidney Dis.2007 December; 50(6):1009-19).

Masson et al. investigate the combination of several circulatingbiomarkers such as NT-proBNP with abnormal midwall fractional shortening(Circulation. Nov. 25, 2014; 130: A12358 Abstract 12358: Abnormal LeftVentricular Midwall Fractional Shortening and Elevated CirculatingBiomarkers Predict High Mortality in Elderly Individuals in the GeneralPopulation).

BRIEF SUMMARY OF THE DISCLOSURE

The present invention relates to a method for predicting the risk of asubject of rapidly progressing to chronic heart failure and/or ofhospitalization due to chronic heart failure and/or death. The method isbased on the determination of at least one biomarker selected from thegroup consisting of a BNP-type peptide, IGFBP7 (IGF binding protein 7),a cardiac Troponin, soluble ST2 (sST2), FGF-23 (Fibroblast Growth Factor23), Growth Differentiation Factor 15 (GDF-15), Intercellular AdhesionMolecule 1 (ICAM-1), and Angiopoietin-2 (ANG2) in a sample of a subject.The method may further encompass the assessment of the presence orabsence of (i) abnormal midwall fractional shortening or (ii) leftventricular hypertrophy. Further envisaged by the present invention aredevices adapted to carry out the present invention.

BRIEF DESCRIPTION OF THE FIGURES

The figures show:

FIG. 1 Incidence for death stratified according to biomarker group(NTproBNP, MFS) in all patients.

FIG. 2 Incidence for death stratified according to biomarker group (cTnTMFS) in all patients.

FIG. 3 Incidence for death stratified according to biomarker group(IGFBP7, MFS) in all patients.

FIG. 4 Incidence for death stratified according to biomarker group(sST2, MFS) in all patients.

FIG. 5 Incidence for death stratified according to biomarker group(FGF23, MFS) in all patients.

FIG. 6 Incidence for death stratified according to biomarker group(NTproBNP MFS) in patients with normal LV mass.

FIG. 7 Incidence for death stratified according to biomarker group(cTnT, MFS) in patients with normal LV mass.

FIG. 8 Incidence for death stratified according to biomarker group(IGFBP7, MFS) in patients with normal LV mass.

FIG. 9 Incidence for death stratified according to biomarker group(sST2, MFS) in patients with normal LV mass.

FIG. 10 Incidence for death stratified according to biomarker group(FGF23, MFS) in patients with normal LV mass.

FIG. 11 Incidence for death stratified according to biomarker group(NTproBNP, IGFBP7, LVH) in all patients.

FIG. 12 Incidence for death stratified according to biomarker group(NTproBNP, IGFBP7, MFS) in all patients.

DETAILED DESCRIPTION OF THE DISCLOSURE

Advantageously, it has been shown in the context of the studiesunderlying the present invention that the measurement of the level of atleast one biomarker selected from the group consisting of a BNP-typepeptide, IGFBP7 (IGF binding protein 7), a cardiac Troponin, soluble ST2(sST2), FGF-23 (Fibroblast Growth Factor 23), Growth DifferentiationFactor 15 (GDF-15), Intercellular

Adhesion Molecule 1 (ICAM-1), and Angiopoietin-2 (ANG2) in a sample of asubject, in combination with the assessment whether the subject suffersfrom abnormal MFS, or not, allows for a reliable identification ofsubjects who are at risk of rapidly progressing to chronic heart failureand/or who are at risk of hospitalization due to chronic heart failureand/or death. It has been further shown in the context of the studiesunderlying the present invention that the measurement of the level of atleast one biomarker selected from the group consisting of IGFBP7 (IGFbinding protein 7), soluble ST2 (sST2), FGF-23 (Fibroblast Growth Factor23), GDF15 (Growth Differentiation Factor15) in a sample of a subject,in combination with the assessment whether the subject suffers from LVH,or not, allows for a reliable identification of subjects who are at riskof rapidly progressing to chronic heart failure and/or who are at riskof hospitalization due to chronic heart failure and/or death.

Accordingly, the present invention relates to a method for predictingthe risk of a subject of rapidly progressing to chronic heart failureand/or of hospitalization due to chronic heart failure and/or death,said method comprising:

-   -   (a) assessing in said subject        -   (i) the presence or the absence of abnormal midwall            fractional shortening (abnormal MFS), and/or        -   (ii) the presence or absence of left ventricular hypertrophy            (LVH),    -   (b) measuring the level of at least one biomarker selected from        the group consisting of a BNP-type peptide, IGFBP7 (IGF binding        protein 7), a cardiac Troponin, soluble ST2 (sST2), FGF-23        (Fibroblast Growth Factor 23), Growth

Differentiation Factor 15 (GDF-15), Intercellular Adhesion Molecule 1(ICAM-1), and Angiopoietin-2 (ANG2) in a sample from the subject, and

-   -   (c) comparing the level(s) of said at least one biomarker to a        reference level (reference levels).

Preferably, the risk is predicted by carrying out the further step d) ofpredicting the risk, or of providing a prediction of the risk of thesubject of rapidly progressing to chronic heart failure and/or ofhospitalization due to chronic heart failure and/or of death. Said stepis based on the results of steps a) b) and c).

The method of the present invention, preferably, is an ex vivo or invitro method. Moreover, it may comprise steps in addition to thoseexplicitly mentioned above. For example, further steps may relate tosample pre-treatments or evaluation of the results obtained by themethod. The method may be carried out manually or assisted byautomation. Preferably, the measurement steps, the calculation steps andthe comparison step may in total or in part be assisted by automation,e.g., by a suitable robotic and sensory equipment for the measurement, acomputer-implemented calculation algorithm on a data processing devicein the calculation steps, or comparison and/or diagnosis algorithm on adata processing device in the comparison step.

In accordance with the present invention, the risk of a subject ofrapidly progressing to chronic heart failure and/or of hospitalizationdue to chronic heart failure and/or of death shall be predicted. Thus, asubject can be identified who is at risk thereof, or who is not at riskthereof. The term “predicting the risk” as used herein, preferably,refers to assessing the probability according to which a subject willrapidly progress to chronic heart failure and/or to assessing theprobability of hospitalization due to chronic heart failure and/or ofdeath. More preferably, the risk/probability within a certain timewindow is predicted. In a preferred embodiment of the present invention,the predictive window, preferably, is an interval of at least 1 year, atleast 2 years, at least 3 years, at least 4 years, at least 5 years, orat least 10 years, or any intermitting time range. In a particularpreferred embodiment of the present invention, the predictive window,preferably, is an interval of 5 years, more preferably of 4 years, ormost preferably, of 3 years. In another preferred embodiment of thepresent invention, the predictive window will be the entire life span ofthe subject. Preferably, said predictive window is calculated from thetime point at which the sample to be tested has been obtained.

As will be understood by those skilled in the art, such a prediction isusually not intended to be correct for 100% of the subjects. The term,however, requires that prediction can be made for a statisticallysignificant portion of subjects in a proper and correct manner. Whethera portion is statistically significant can be determined without furtherado by the person skilled in the art using various well known statisticevaluation tools, e.g., determination of confidence intervals, p-valuedetermination, Student's t-test, Mann-Whitney test etc. Details arefound in Dowdy and Wearden, Statistics for Research, John Wiley & Sons,New York 1983. Preferred confidence intervals are at least 90%, at least95%, at least 97%, at least 98%, or at least 99%. The p-values are,preferably, 0.1, 0.05, 0.01, 0.005, or 0.0001. Preferably, theprobability envisaged by the present invention allows that theprediction of an increased, normal or decreased risk will be correct forat least 60%, at least 70%, at least 80%, or at least 90% of thesubjects of a given cohort or population. The term, preferably, relatesto predicting whether a subject is at elevated risk or reduced risk ascompared to the average risk in a population of subjects.

The term “predicting the risk” as used herein means that the subject tobe analyzed by the method of the present invention is allocated eitherinto the group of subjects being at risk of rapidly progressing tochronic heart failure and/or of hospitalization due to chronic heartfailure and/or death, or into the group of subjects being not at risk ofrapidly progressing to chronic heart failure and/or of hospitalizationdue to chronic heart failure and/or death. At subject who is at risk,preferably, is a subject who is at elevated risk of rapidly progressingto chronic heart failure and/or of hospitalization due to chronic heartfailure and/or death (in particular within the predictive window).Preferably, said risk is elevated as compared to the average risk in acohort of subjects (i.e. a group of subjects). At subject who is not atrisk, preferably, is a subject who is at reduced risk of rapidlyprogressing to chronic heart failure and/or of hospitalization due tochronic heart failure and/or death (in particular within the predictivewindow). Preferably, said risk is reduced as compared to the averagerisk in a cohort of subjects (i.e. a group of subjects). Accordingly,the method of the present invention allows for differentiating betweenan elevated risk or a reduced risk. A subject who is at risk ofpreferably has a risk of 12% or larger, or, more preferably of 15% orlarger, or most preferably of 20% or larger of rapidly progressing tochronic heart failure and/or of hospitalization due to chronic heartfailure and/or death, preferably, within a predictive window of 3 or 4years. A subject who is not at risk preferably has a risk of lower than10%, more preferably of lower than, 8%, or most preferably of lower than7% of rapidly progressing to chronic heart failure and/or ofhospitalization due to chronic heart failure and/or death, preferably,within a predictive window of 3 or 4 years.

In an embodiment, the risk of hospitalization is predicted. Theexpression “hospitalization” as used herein, preferably, means that thesubject is admitted to a hospital, in particular on an in-patient basis.The hospitalization should be due to chronic heart failure. Thus,chronic heart failure shall be the cause for the hospitalization. In anembodiment, the risk of the subject of rapidly progressing to chronicheart failure is predicted. The expression “rapidly progressing tochronic heart failure” is well understood by the skilled person. Asubject who progresses rapidly to chronic heart failure, preferably, isa subject who progresses to chronic heart failure within a window periodas described elsewhere herein. Chronic heart failure preferably meansheart failure classified as stage C or D according to according to theACC/AHA classification (for the ACC/AHA classification, see elsewhereherein). If the subject to be tested has heart failure classified asstage A according to according to the ACC/AHA classification (or if thesubject does not suffer from heart failure), the term “chronic heartfailure” may mean heart failure classified as stage B, C or D.

In an embodiment, the risk of death is predicted. The term “death” asused herein preferably relates to death from any cause, and, morepreferably, to death from cardiac cause, and most preferably to deathdue to heart failure.

The phrase “providing a prediction” as used herein refers to using theinformation or data generated relating to the level a biomarker asreferred to herein in a sample of a subject, and relating to thepresence of absence of (i) abnormal MFS or (ii) LVH to predict a risk asreferred to herein. The information or data may be in any form, written,oral or electronic. In some embodiments, using the information or datagenerated includes communicating, presenting, reporting, storing,sending, transferring, supplying, transmitting, dispensing, orcombinations thereof. In some embodiments, communicating, presenting,reporting, storing, sending, transferring, supplying, transmitting,dispensing, or combinations thereof are performed by a computing device,analyzer unit or combination thereof. In some further embodiments,communicating, presenting, reporting, storing, sending, transferring,supplying, transmitting, dispensing, or combinations thereof areperformed by a laboratory or medical professional. In some embodiments,the information or data includes a comparison of the level of thebiomarker(s) to a reference level (or levels). In some embodiments, theinformation or data includes an indication that the subject is diagnosedto be at risk.

The “subject” as referred to herein is, preferably, a mammal. Mammalsinclude, but are not limited to, domesticated animals (e.g., cows,sheep, cats, dogs, and horses), primates (e.g., humans and non-humanprimates such as monkeys), rabbits, and rodents (e.g., mice and rats).Preferably, the subject is a human subject. Preferably, the subject is65 years or older, more preferably, the subject is 75 years old orolder, most preferably, the subject is 80 years old or older. The terms“subject” and “patient” are used interchangeably herein.

In a preferred embodiment of the present invention, the subject isapparently healthy with respect to heart failure. A subject who isapparently healthy with respect to heart failure preferably does notshow symptoms of heart failure (and thus is asymptomatic with respect toheart failure). A subject who does not show symptoms of heart failurepreferably has no limitation of physical activity, and ordinary physicalactivity results in undue breathlessness, fatigue, or palpitations.However, it is envisaged that subject has heart failure classified asstage A or B according to the ACC/AHA classification, in particularheart failure classified as stage A, or early stage B according to theACC/AHA classification.

The ACC/AHA classification is a classification for heart failuredeveloped by the American College of Cardiology and the American HeartAssociation (for the classification, see J. Am. Coll. Cardiol. 2001; 38;2101-2113, updated in 2005, see J. Am. Coll. Cardiol. 2005; 46; e1-e82which is herewith incorporated by reference in its entirety). Theclassification is also described in Mureddu et al., European Journal ofHeart Failure (2012) 14, 718-729 which is herewith incorporated byreference as well. 4 stages A, B, C and D are defined. Stages A and Bare not HF (heart failure) but are considered to help identify patientsearly before developing “truly” HF. Stages A and B patients are bestdefined as those with risk factors for the development of HF. Forexample, patients with coronary artery disease, hypertension, ordiabetes mellitus who do not yet demonstrate impaired left ventricular(LV) function, hypertrophy, or geometric chamber distortion would beconsidered stage A, whereas patients who are asymptomatic butdemonstrate impaired LV function would be designated as stage B. Stage Cthen denotes patients with current or past symptoms of HF associatedwith underlying structural heart disease (the bulk of patients with HF),and stage D designates patients with truly refractory HF. The ACC/AHAclassification and the term “heart failure” are also explained in WO2012/025355 which herewith is incorporated by reference in its entirety.

Preferably, the subject in the context of the present invention does nothave impaired renal function. Preferably, the subject shall not sufferfrom renal failure, in particular the subject shall not suffer fromacute, chronic and/or end stage renal failure. Thus, the subjectpreferably is preferably not a hemodialyzed subject.

How to assess whether a subject exhibits impaired renal function is wellknown in the art. Renal disorders can be diagnosed by any means knownand deemed appropriate. Particularly, renal function can be assessed bymeans of the glomerular filtration rate (GFR). For example, the GFR maybe calculated by the Cockgroft-Gault or the MDRD formula (Levey 1999,Annals of Internal Medicine, 461-470). GFR is the volume of fluidfiltered from the renal glomerular capillaries into the Bowman's capsuleper unit time. Clinically, this is often used to determine renalfunction. All calculations derived from formulas such as the CockgroftGault formula of the MDRD formula deliver estimates and not the “real”GFR) by injecting inulin into the plasma. Since inulin is not reabsorbedby the kidney after glomerular filtration, its rate of excretion isdirectly proportional to the rate of filtration of water and solutesacross the glomerular filter. In clinical practice however, creatinineclearance is used to measure GFR. Creatinine is an endogenous molecule,synthesized in the body, which is freely filtered by the glomerulus (butalso secreted by the renal tubules in very small amounts). Creatinineclearance (CrCl) is therefore a close approximation of the GFR. The GFRis typically recorded in milliliters per minute (mL/min). The normalrange of GFR for males is 97 to 137 mL/min, the normal range of GFR forfemales is 88 to 128 ml/min. Thus, it is particularly contemplated thatthe GFR of a subject who does not exhibit impaired renal function iswithin this range. Moreover, said subject preferably, has a bloodcreatinine level (in particular a serum creatinine level) of lower than0.9 mg/dl, more preferably of lower than 1.1 mg/dl and most preferablyof lower than 1.3 mg/dl.

Step (a) of the aforementioned method of the present invention comprisestwo alternative embodiments: (i) and (ii). According to alternative (i),the presence or the absence of abnormal midwall fractional shortening(abnormal MFS) is assessed. According to alternative (ii), the presenceor absence of left ventricular hypertrophy (LVH) is assessed.

It has been shown in the context of the methods of the presentinvention, that the risk of the subject can be predicted even before thesubject suffers from left ventricular hypertrophy (embodiment (i)).However, the risk can be also reliably predicted, if the subject suffersfrom LVH (embodiment (ii)).

If embodiment (i) of step (a) of the aforementioned method is carriedout, the subject preferably does not suffer from LVH. The term “leftventricular hypertrophy” is well known in the art. A detailed overviewon left ventricular hypertrophy can be, e.g. found in standard textbooks (see Swamy Curr Cardiol Rep (2010) 12:277-282). LVH can bedetected by electrocardiography, echocardiography, or cardiac magneticresonance imaging (MRI). Preferably, LVH is detected byechocardiography. Moreover, criteria for the diagnosis of LVH are wellknown in the art (Mancia et al., European Heart J. 2007, 28: 1462, DieInnere Medizin: Referenzwerk für den Facharzt-Wolfgang Gerok-2007, page293, Swamy Curr Cardiol Rep (2010) 12:277-282). The term “leftventricular hypertrophy” (abbreviated “LVH”) as used herein, preferably,relates to a thickening of the walls of the ventricles. LVH is,preferably, a response to a chronically increased workload on the heart.LVH is found in patients suffering from arterial hypertension is adisease requiring treatment. In the context of the present invention therisk is predicted before the subject suffers from LVH. Accordingly, thesubject to be tested preferably does not suffer from LVH. A subject whodoes not suffer from LVH, preferably, has a normal left ventricularmass.

However, it is also envisaged that the subject may suffer from LVH, ifembodiment (i) of step (a) of the aforementioned method is carried out.

The diagnosis of LVH and thus the assessment of the left ventricularmass, preferably, includes measurements of the septum diameter, leftventricular posterial wall thickness and end diastolic diameter, withcalculation of left ventricular mass according to formulae known in theart. Particularly preferred criteria for diagnosing LVH are e.g.disclosed in the guidelines (Mancia et al., European Heart J. 2007, 28:1462). Preferably, the Cornell voltage criteria, the Cornell productcriteria, the Sokolow-Lyon voltage criteria or the Romhilt-Estes pointscore system is/are used for the diagnosis of LVH and thus for theassessment of the left ventricular mass (see e.g. Mancia et al.,European Heart J. 2007, 28: 1462).

If the subject is male, the following applies: a subject is consideredto have a normal left ventricular mass (and thus does not suffer fromLVH), if the left ventricular mass index of the male subject is,preferably, equal to or lower than 105 g/m², or, more preferably, isequal to or lower than 110 g/m², or, most preferably, is equal to orlower than 115 g/m². If the subject is female, the following applies: asubject is considered to have a normal left ventricular mass, if theleft ventricular mass index of the subject is, preferably, equal to orlower than 85 g/m², or, more preferably, is equal to or lower than 90g/m², or, most preferably, is equal to or lower than 96 g/m². It is tobe understood that a subject who has a normal left ventricular mass doesnot suffer from LVH (see, e.g. Drazner M H, Dries D L, Peshock R M,Cooper R S, Klassen C, Kazi F, Willett D, Victor R G. Left ventricularhypertrophy is more prevalent in blacks than whites in the generalpopulation: the Dallas Heart Study. Hypertension. 2005; 46:124-129).

In step (a), embodiment (i), of the method of the present invention, thepresence or the absence of abnormal midwall fractional shortening(abnormal MFS) shall be assessed. Thus, it is assessed whether thesubject suffers from abnormal MFS, or not. A subject who does not sufferfrom abnormal MFS has a normal MFS.

The expression “midwall fractional shortening” is well known in art(abbreviated herein as “MFS”). MFS is an early sign of LV dysfunction isreduced LV midwall fractional shortening. Preferably, midwall fractionalshortening is considered as abnormal, if it is lower than 15%. Thiscut-off point has been used as a reference value in the setting of HFand has demonstrated prognostic relevance in hypertensive subjects (seeMurredu et al., European Journal of Heart Failure (2012) 14, 718-729).Also preferably, midwall fractional shortening is considered asabnormal, if it is lower than 14%, or 13%. Further, midwall fractionalshortening is considered as normal, if it is larger than (or equal to)15%. Also preferably, midwall fractional shortening is considered asnormal, if it is larger than 16%, or 17%.

How to determine midwall fractional shortening (or LVH) and thus how toassess the presence or the absence of abnormal midwall fractionalshortening (or LVH) in a subject is well known in the art. Preferably,the assessment is based on echocardiographic images of the heartobtained from the subject to be tested. The images shall allow forassessing the presence or absence of abnormal midwall fractionalshortening (or LVH). They may be obtained by any echocardiographytechnique deemed appropriate, in particular M-mode echocardiography, 2Dspeckle tracking echocardiography, Doppler echocardiography, ortwo-dimensional (2D) echocardiography.

In an embodiment, midwall fractional shortening may be calculated fromthe two-shell cylindrical model as described by Shimizu et al. which isherewith incorporated by reference with respect to its entire disclosurecontent (Shimizu G, Hirota Y, Kita Y, Kawamura K, Saito T, Gaasch W H.Left ventricular midwall mechanics in systemic arterial hypertension.Circulation 1991;83:1676-84)). This method is a refinement of theconventional midwall method and provides data that reflect shortening ofa theoretic circumferential midwall fiber or ring of myocardium. Itassumes a constant left ventricular mass throughout the cardiac cycleand does not require the assumption that inner and outer wall thickeningfractions are equal. The determination of MFS is also described by Mayetet al. or in earlier papers from Shimizu et al. all of which areherewith incorporated by reference. (see e.g. Mayet et al.,Hypertension. 2000; 36:755-759; Shimizu G, Zile M R, Blaustein A S,Gaasch W H. Left ventricular chamber filling and midwall fiberlengthening in patients with left ventricular hypertrophy:overestimation of fiber velocities by conventional midwall measurements.Circulation. 1985;71:266-272, or Shimizu G, Conrad C H, Gaasch W H.Phase-plane analysis of left ventricular chamber filling and midwallfiber lengthening in patients with left ventricular hypertrophy.Circulation. 1987; 75(suppl I):I-34-I-39). Moreover, the determinationof MFS has been described by de Simone et al. (JACC, 1994, Vol. 23(6):1444-51) which is also incorporated by reference in its entirety. Inanother embodiment, MFS is assessed as described by Mureddu et al.(European Journal of Heart Failure (2012) 14, 718-729). Preferably, MFSas used herein is determined according to the method as described byShimizu in 1987 or 1985, more preferably MFS, is determined according toMayet et al., even more preferably, MFS is determined according to deSimone, and most preferably, MFS is determined as described by Mureddu.

As further described in the Examples section, the measurement of thelevel of at least one biomarker as referred to herein is in particularadvantageous in subjects who suffer from abnormal MFS (see alsoFigures). In subjects with abnormal MFS, the measurement of the at leastone biomarker allows for a very reliable differentiation between asubject who is at risk and who is not risk. The results in the Examplessection show that a subject might be have a very low risk, although hesuffers from abnormal midwall fractional shortening. However, there arealso subjects who suffer from abnormal MFS and who have a very highrisk. The determination of at least one biomarker as referred to hereinallows to identify those subjects who have a high risk and a low risk.

The discriminatory power of some biomarkers as referred to herein (inparticular of a BNP-type peptide, in particular NT-proBNP, sST2, IGFBP7,a cardiac Troponin, in particular cTnT, and/or FGF23 is particularlyadvantageous in subjects who have a normal ventricular mass but anabnormal MFS. Thus, if embodiment (i) of step (a) is carried out, the atleast one biomarker is preferably, a BNP-type peptide, sST2, IGFBP7, acardiac Troponin and/or FGF23. Preferably, the marker is sST2, morepreferably, the marker is IGFBP7, most preferably, the marker isNT-proBNP. In addition also preferred that the marker is Troponin T orFGF23. It is to be understood that the biomarker GDF-15 and/or may bedetermined as well.

In an embodiment, step b) may be carried out after the presence orabsence of abnormal MFS has been assessed. In particular, step b) maycarried out in a subject who suffers from abnormal MFS. Accordingly, themethod of the present in invention may further comprises step a1) ofselecting a subject who suffers from abnormal midwall fractionalshortening based on the assessment in step a). Thus, the level of the atleast biomarker is measured in a subject suffering from abnormal MFS.

Alternatively, the level of the at least one biomarker may be measuredas a first step. Afterwards, the presence of abnormal midwall fractionalshortening is assessed.

In step (a), embodiment (ii), of the method of the present invention,the presence or the absence of LVH shall be assessed. Thus, it isassessed whether the subject suffers from LVH, or not. A subject whodoes not suffer from LVH has a normal left ventricular mass (as outlinedelsewhere herein.

If embodiment (ii) of step (a) of the aforementioned method is carriedout, the at least one biomarker is preferably selected from IGFBP7 (IGFbinding protein 7), soluble ST2 (sST2), FGF-23 (Fibroblast Growth Factor23), and GDF15 (Growth Differentiation Factor15). In particular, the atleast one biomarker is IGFBP-7 and/or sST2. However, the remainingmarkers such as NT-proBNP may be determined as well. Preferredcombinations of biomarkers (patient's characteristics) are as follows:

-   -   IGFBP7+a BNP-type peptide, in particular NTproBNP    -   ST2+a BNP-type peptide, in particular NTproBNP    -   FGF23+a BNP-type peptide, in particular NTproBNP    -   IGFBP7+ST2    -   age+a BNP-type peptide, in particular NTproBNP    -   age+NTproBNP+cTn, in particular hs-cTnT

“Age” preferably means that the subject to be tested is 65 years orolder

Another preferred combination is a BNP-type peptide, in particularNTproBNP, and IGFBP7, in particular in aged subjects (in particular asubject is 65 years or older), preferably in combination with theassessment of the presence or absence of left ventricular hypertrophy(LVH).

Another preferred combination is a BNP-type peptide, in particularNTproBNP, and IGFBP7, in particular in aged subjects (in particular asubject is 65 years or older) in combination with the assessment of thepresence or absence of abnormal MFS.

The term “sample” refers to a sample of a body fluid, to a sample ofseparated cells or to a sample from a tissue or an organ. Samples ofbody fluids can be obtained by well-known techniques and include,samples of blood, plasma, serum, urine, lymphatic fluid, sputum,ascites, or any other bodily secretion or derivative thereof. Tissue ororgan samples may be obtained from any tissue or organ by, e.g., biopsy.In particular, it is envisaged that the sample is a blood, blood serum,or blood plasma sample.

In accordance with the present invention, the level of at least onebiomarker as referred to herein shall be measured. The term “at leastone” means one or more than one. Preferably, the level(s) of one, two,three, four, five, six, seven, or eight biomarkers are measured in thecontext of the present invention.

Moreover, it is envisaged that the level of at least one furtherbiomarker is measured level and that the measured level of the at leastone further biomarker is compared to a reference level. Preferably, theat least one further biomarker is P1GF (Placental Growth Factor). Morepreferably, the at least one further biomarker is MMP2 (matrixmetalloproteinase-2). MMP2 (also known as 72 kDa type IV collagenase andgelatinase A) is an enzyme that in humans is encoded by the MMP2 gene(for the sequence of human MMP2, see e.g. Uniprot (P08253).

In addition, it is envisaged to assess a further characteristic orfurther characteristics of the subject for the assessment of the risk,preferably age or gender (sex), in particular age and gender (sex).Preferably, the risk is predicted based on the comparison step and basedon the patient's characteristic(s) (such as age and sex). Morepreferably, the risk is predicted based on the comparison step, based onthe presence or absence of abnormal MFS and/or LVH (in particular, basedon the presence of MFS and/or LVH), and based on the patient'scharacteristic(s) (such as age and sex).

In a preferred embodiment, the levels of biomarkers are measured incombination. Thus, more than one level is measured. Moreover,combinations of a biomarker (or of biomarkers) with a patient'scharacteristic (patient's characteristics) are envisaged. Preferredcombinations are shown in FIG. 11 and FIG. 12 and, in particular inTable 1 of the Examples section, in particular, the combination of aBNP-type peptide, in particular NTproBNP and IGFBP7 with the assessmentof LVH or MFS, respectively, is preferred. Further preferredcombinations are shown in Table 2 of the Examples section.

Further preferred combinations of biomarkers (and patient'scharacteristics) are:

-   -   a BNP-type peptide, in particular NTproBNP, and a cardiac        Troponin, in particular cTnT    -   IGFBP7 and a BNP-type peptide, in particular NT-proBNP    -   IGFBP7 and ST2    -   ST2 and a BNP-type peptide, in particular NT-proBNP    -   GDF15 and a BNP-type peptide, in particular NT-proBNP    -   FGF23 and a BNP-type peptide, in particular NT-proBNP    -   Age and a BNP-type peptide, in particular NT-proBNP    -   Age, a cardiac Troponin, in particular cTnT (Troponin T) and a        BNP-type peptide, in particular NT-proBNP

Further preferred combinations are: at least one biomarker selected frombiomarker selected from a cardiac Troponin, in particular TnT, FGF23, aBNP-type peptide, in particular NTproBNP, IGFBP7, sST2, and GDF15, incombination with age and/or (in particular and) gender.

The term “measuring” (herein also referred to a “determining”) the levelof a marker as referred to herein refers to the quantification of thebiomarker, e.g. to determining the level of the biomarker in the sample,employing appropriate methods.

The biomarkers to be measured in connection with the present inventionare protein biomarkers. How to measure the level, and thus how todetermine the amount of a protein biomarker is well know in the art andis e.g. described in WO 2014/040759 which is herewith incorporated byreference in its entirety, see in particular page 15, line 15, to page19 line 25.

In an embodiment, the level of the at least one biomarker is measured bycontacting the sample with a binding agent that specifically binds tothe respective marker, thereby forming a complex between the agent andsaid marker, detecting the level of complex formed, and therebymeasuring the level of said marker.

Preferably, the binding agent binds specifically to a biomarker asreferred to herein. Preferred binding agents include antibodies, nucleicacids, peptides or polypeptides such as receptors or binding partnersfor the peptide or polypeptide and fragments thereof comprising thebinding domains for the peptides, and aptamers, e.g. nucleic acid orpeptide aptamers, in particular antibodies. Antibodies as referred toherein include both polyclonal and monoclonal antibodies, as well asfragments thereof, such as Fv, Fab and F(ab)2 fragments that are capableof binding antigen or hapten. The present invention also includes singlechain antibodies and humanized hybrid antibodies wherein amino acidsequences of a non-human donor antibody exhibiting a desiredantigen-specificity are combined with sequences of a human acceptorantibody. The term “specific binding” or “specifically bind” refers to abinding reaction wherein binding pair molecules exhibit a binding toeach other under conditions where they do not significantly bind toother molecules. The term “specific binding” or “specifically binds”,when referring to a protein or peptide as biomarker, refers to a bindingreaction wherein a binding agent binds to the corresponding biomarkerwith an affinity of at least 10⁻⁷ M. The term “specific binding” or“specifically binds” preferably refers to an affinity of at least 10⁻⁸ Mor even more preferred of at least 10⁻⁹ M for its target molecule. Theterm “specific” or “specifically” is used to indicate that othermolecules present in the sample do not significantly bind to the bindingagent specific for the target molecule. Preferably, the level of bindingto a molecule other than the target molecule results in a bindingaffinity which is only 10% or less, more preferably only 5% or less ofthe affinity to the target molecule.

Third, the binding agent may be coupled covalently or non-covalently toa label allowing detection and measurement of the binding agent.Labeling may be done by direct or indirect methods. Direct labelinginvolves coupling of the label directly (covalently or non-covalently)to the binding agent. Indirect labeling involves binding (covalently ornon-covalently) of a secondary binding agent to the first binding agent.The secondary binding agent should specifically bind to the firstbinding agent. Said secondary binding agent may be coupled with asuitable label and/or be the target (receptor) of tertiary binding agentbinding to the secondary binding agent.

The biomarkers as referred to herein are well known in the art.

The Brain Natriuretic Peptid type peptide (herein also referred to asBNP-type peptide) is preferably selected from the group consisting ofpre-proBNP, proBNP, NT-proBNP, and BNP. The pre-pro peptide (134 aminoacids in the case of pre-proBNP) comprises a short signal peptide, whichis enzymatically cleaved off to release the pro peptide (108 amino acidsin the case of proBNP). The pro peptide is further cleaved into anN-terminal pro peptide (NT-pro peptide, 76 amino acids in case ofNT-proBNP) and the active hormone (32 amino acids in the case of BNP).Preferably, brain natriuretic peptides according to the presentinvention are NT-proBNP, BNP, and variants thereof. BNP is the activehormone and has a shorter half-life than its respective inactivecounterpart NT-proBNP. Preferably, the Brain Natriuretic Peptid-typepeptide is BNP (Brain natriuretic peptide), and more preferablyNT-proBNP (N-terminal of the prohormone brain natriuretic peptide).

The term “cardiac Troponin” refers to all Troponin isoforms expressed incells of the heart and, preferably, the subendocardial cells. Theseisoforms are well characterized in the art as described, e.g., inAnderson 1995, Circulation Research, vol. 76, no. 4: 681-686 andFerrieres 1998, Clinical Chemistry, 44: 487-493. Preferably, cardiacTroponin refers to Troponin T and/or Troponin I, and, most preferably,to Troponin T.

IGF binding protein 7 (=IGFBP7) is a 30-kDa modular glycoprotein knownto be secreted by endothelial cells, vascular smooth muscle cells,fibroblasts, and epithelial cells (Ono, Y., et al., Biochem Biophys ResComm 202 (1994) 1490-1496). Preferably, the term “IGFBP7” refers tohuman IGFBP7. The sequence of the protein is well known in the art andis e.g. accessible via GenBank (NP_001240764.1).

ST2, also known as “Interleukin 1 receptor-like 1” is a member of theIL-1 receptor family that is produced by cardiac fibroblasts andcardiomyocytes under conditions of mechanical stress. ST2 is aninterleukin-1 receptor family member and exists in both membrane-boundisoform and a soluble isoform (sST2). In the context of the presentinvention, the amount of soluble ST2 shall be determined (see Dieplingeret al. (Clinical Biochemistry, 43, 2010: 1169 to 1170). ST2 also knownas Interleukin 1 receptor-like 1 or IL1RL1, is encoded in humans by theIL1RL1 gene. The sequence of the human ST2 polypeptide is well known inthe art, and e.g. accessible via GenBank, see NP_003847.2 GI:27894328.

The biomarker fibroblast growth factor-23 (abbreviated “FGF-23”) is wellknown in the art. FGF-23 a key player in the regulation ofcalcium-phosphate and vitamin D metabolism and has a causal role in thepathogenesis of LV hypertrophy, a major determinant of cardiovascularevents. Preferably, FGF-23 is human FGF-23. The sequence of human FGF-23is well known in the art, e.g. the amino sequence can be assessed viaGenBank accession number NM_020638.1 GI:10190673. Moreover, the sequenceis also disclosed in Shimada et al., 2001, PNAS, vol. 98(11) page 6500to 6505.

The term “Growth-Differentiation Factor-15” or “GDF-15” relates to apolypeptide being a member of the transforming growth factor (TGF)cytokine superfamily. The terms polypeptide, peptide and protein areused interchangeable throughout this specification. GDF-15 wasoriginally cloned as macrophage-inhibitory cytokine 1 and later alsoidentified as placental transforming growth factor-15, placental bonemorphogenetic protein, non-steroidal anti-inflammatory drug-activatedgene 1, and prostate-derived factor (Bootcov loc cit; Hromas, 1997Biochim Biophys Acta 1354:40-44; Lawton 1997, Gene 203:17-26;Yokoyama-Kobayashi 1997, J Biochem (Tokyo), 122:622-626; Paralkar 1998,J Biol Chem 273:13760-13767). Amino acid sequences for GDF-15 aredisclosed in WO99/06445, WO00/70051, WO2005/113585, Bottner 1999, Gene237: 105-111, Bootcov loc. cit, Tan loc. cit., Baek 2001, Mol Pharmacol59: 901-908, Hromas loc cit, Paralkar loc cit, Morrish 1996, Placenta17:431-441.

Intercellular adhesion molecule-1 (ICAM-1; frequently also referred toas CD54) is a transmembrane glycoprotein which is typically expressed onendothelial cells and cells of the immune system. The structure ofICAM-1 is characterized by heavy glycosylation and consists of anextracellular portion, which forms five immunoglobulin (Ig)-likedomains. These domains are attached to a single transmembrane region anda short cytoplasmic tail. Several ligands for ICAM-1 have beendescribed; it binds to integrins of type CD11a/CD18, or CD11b/CD18 andis also exploited by Rhinovirus as a receptor. The amino acid sequenceof human ICAM-1, preferably, is given in UniProt Entry P05362. TheICAM-1 referred to in accordance with the present invention furtherencompasses allelic and other variants of said specific sequence.

The marker “Angiopoietin 2” (ANG2) is well known in the art (see e.g.Sarah Y. Yuan; Robert R. Rigor (30 Sep. 2010). Regulation of EndothelialBarrier Function. Morgan & Claypool Publishers. ISBN 978-1-61504-120-6).Angiopoietin 2 is part of a family of vascular growth factors that playa role in embryonic and postnatal angiogenesis. Angiopoietin-2 isproduced and stored in Weibel-Palade bodies in endothelial cells andacts as a TEK tyrosine kinase antagonist. angiopoietin-2 is a marker forearly cardiovascular disease in children on chronic dialysis (Shroff etal. (2013). “Circulating angiopoietin-2 is a marker for earlycardiovascular disease in children on chronic dialysis.”. PLoS ONE 8(2): e56273). The sequence of ANG2 is e.g. accessible via UniProt (seeAccession Number O15123)

The term “level” as used herein encompasses the absolute amount of abiomarker as referred to herein, the relative amount or concentration ofthe said biomarker as well as any value or parameter which correlatesthereto or can be derived therefrom.

The term “comparing” as used herein refers to comparing the level of thebiomarker in the sample from the individual or patient with thereference level of the biomarker specified elsewhere in thisdescription. It is to be understood that comparing as used hereinusually refers to a comparison of corresponding parameters or values,e.g., an absolute amount is compared to an absolute reference amountwhile a concentration is compared to a reference concentration or anintensity signal obtained from the biomarker in a sample is compared tothe same type of intensity signal obtained from a reference sample. Thecomparison may be carried out manually or computer assisted. The valueof the measured or detected level of the biomarker in the sample fromthe individual or patient and the reference level can be, e.g., comparedto each other and the said comparison can be automatically carried outby a computer program executing an algorithm for the comparison.

The term “reference level” is well known in the art. Preferred referencelevels can be determined by the skilled person without further ado.Preferably, the term “reference level” herein refers to a predeterminedvalue for the respective biomarker. In this context “level” encompassesthe absolute amount, the relative amount or concentration as well as anyvalue or parameter which correlates thereto or can be derived therefrom.Preferably, the reference level is a level which allows for allocatingthe subject into a group of subjects who are at risk of rapidlyprogressing to chronic heart failure and/or of hospitalization due tochronic heart failure and/or death, or into a group of subjects who arenot at risk of rapidly progressing to chronic heart failure and/or ofhospitalization due to chronic heart failure and/or death. Thus, thereference level shall allow for differentiating between a subject who isat risk or who is not at risk (of rapidly progressing to chronic heartfailure and/or of hospitalization due to chronic heart failure) and/ordeath.

As the skilled artisan will appreciate the reference level ispredetermined and set to meet routine requirements in terms of e.g.specificity and/or sensitivity. These requirements can vary, e.g. fromregulatory body to regulatory body. It may for example be that assaysensitivity or specificity, respectively, has to be set to certainlimits, e.g. 80%, 90%, 95% or 98%, respectively.

These requirements may also be defined in terms of positive or negativepredictive values. Nonetheless, based on the teaching given in thepresent invention it will always be possible for a skilled artisan toarrive at the reference level meeting those requirements. In oneembodiment the reference level is determined in a reference sample orsamples from a patient (or group of patients) who are at risk. Inanother embodiment, the referenced is determined in a reference sampleor samples from a patient (or group of patients) who are not at risk (ofrapidly progressing to chronic heart failure and/or of hospitalizationdue to chronic heart failure and/or death). The reference level in oneembodiment has been predetermined in reference samples from the diseaseentity to which the patient belongs. In certain embodiments thereference level can e.g. be set to any percentage between 25% and 75% ofthe overall distribution of the values in a disease entity investigated.In other embodiments the reference level can e.g. be set to the median,tertiles or quartiles as deter-mined from the overall distribution ofthe values in reference samples from a disease entity investigated. Inone embodiment the reference level is set to the median value asdetermined from the overall distribution of the values in a diseaseentity investigated. The reference level may vary depending on variousphysiological parameters such as age, gender or subpopulation, as wellas on the means used for the determination of the biomarkers referred toherein. In one embodiment, the reference sample is from essentially thesame type of cells, tissue, organ or body fluid source as the samplefrom the individual or patient subjected to the method of the invention,e.g. if according to the invention blood is used as a sample todetermine the level of biomarkers in the individual, the reference levelis also determined in blood or a part thereof.

Preferably, the following applies as algorithm:

Preferably, a level (or levels) of the at least one biomarker above thereference level(s) in indicates that the subject is at risk of rapidlyprogressing to chronic heart failure and/or of hospitalization due tochronic heart failure and/or death. Also preferably, a level (or levels)of the at least one biomarker below the reference level(s) indicatesthat the subject is not at risk of rapidly progressing to chronic heartfailure and/or of hospitalization due to chronic heart failure and/ordeath.

The aforementioned algorithm applies in particular, if the presence ofabnormal MFS or LVH has been assessed in step a) of the method of thepresent invention, i.e. if it has been assessed in step a) that thesubject suffers from abnormal MFS or from LVH.

In certain embodiments, the term “larger than the reference level” or“above the reference level” refers to a level of the biomarker in thesample from the individual or patient above the reference level or to anoverall increase of 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%,85%, 90%, 95%, 100% or greater, determined by the methods describedherein, as compared to the reference level. In certain embodiments, theterm increase refers to the increase in biomarker level in the samplefrom the individual or patient wherein, the increase is at least about1.5-, 1.75-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 15-, 20-, 25-, 30-,40-, 50-, 60-, 70-, 75-, 80-, 90-, or 100-fold higher as compared to thereference level, e.g. predetermined from a reference sample. In certainembodiments, the term “lower than the reference level” or “below” hereinrefers to a level of the biomarker in the sample from the individual orpatient below the reference level or to an overall reduction of 5%, 10%,20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99% or greater, determined by the methods described herein, as comparedto the reference level. In certain embodiments, the term decrease inbiomarker level in the sample from the individual or patient wherein thedecreased level is at most about 0.9-, 0.8-, 0.7-, 0.6-, 0.5-, 0.4-,0.3-, 0.2-, 0.1-, 0.05-, or 0.01-fold of the reference level, e.g.predetermined from a reference sample, or lower.

Preferred reference levels can be determined by the skilled personwithout further ado. The reference level may be age-dependent. However,this is taken into account by the skilled person. For example, thereference level may be the median level in a population of subjects.Preferred reference levels for the markers disclosed herein are shown inthe Examples section in Table 3. Depending on the desired sensitivityand specificity, the reference levels may differ.

In a preferred embodiment of the methods of the present invention, saidmethods further comprise the step of recommending and/or initiating atleast one suitable therapy, if it is predicted that the subject is atrisk of rapidly progressing to chronic heart failure and/or ofhospitalization due to chronic heart failure and/or death. Accordingly,the present invention also pertains the a method of treatment.

Preferably, a The term “therapy” as used in the context of the presentinvention encompasses life style changes, diet regimen, interventions onthe body as well as medicinal treatment, i.e. treatment with amedicament (or with medicaments).

Medicaments suitable for the treatment are well known in the art, seee.g. Heart Disease, 2008, 8th Edition, Eds. Braunwald, ElsevierSounders, chapter 24 (in respect to heart failure) and chapter 41 (inrespect to hypertension). These treatments are a part of the presentinvention.

Preferably, the administration of such medicaments aims to reduce therisk of the subject rapidly progressing to chronic heart failure and/orof hospitalization due to chronic heart failure and/or death.Preferably, the medicament is selected from the group consisting of anangiotensin-converting enzyme (ACE) inhibitors, an angiotensin receptorantagonist (ARB), an aldosterone antagonists, a diuretic and a betablocker. In particular, the medicament is an angiotensin receptorantagonist, or an ACE inhibitor.

Preferred diuretics are loop diuretics, thiazide and thiazide-likediuretics. Preferred beta blockers are proprenolol, metoprolol,bisoprolol, carvedilol, bucindolol, and nebivolol. Preferred ACEinhibitors are Enalapril, Captopril, Ramipril and Trandolapril.Preferred angiotensin receptor antagonists are Losartan, Valsartan,Irbesartan, Candesartan, Telmisartan, and Eprosartan. Preferredaldosterone antagonists like Eplerone, Spironolactone, Canrenone,Mexrenone and Prorenone. Preferred calcium antagonists aredihydropyridines, verapamil, and diltiazem.

Life style changes include smoking cessation, moderation of alcoholconsumption, increased physical activity, weight loss, sodium (salt)restriction, weight management and healthy eating, daily fish oil, saltrestriction.

Further preferred therapies are disclosed by Frohlich et al. (Journal ofHypertension 2011, 29:17-26), which is herewith incorporated byreference with respect to its entire disclosure content. In particular,it is referred to page 21 and 22 of this reference.

The definitions given herein below apply mutatis mutandis to thefollowing embodiments of the present invention.

The present invention also relates to the use of at least one biomarkerselected from the group consisting of a BNP-type peptide, IGFBP7 (IGFbinding protein 7), a cardiac Troponin, soluble ST2 (sST2), FGF-23(Fibroblast Growth Factor 23), Growth Differentiation Factor 15(GDF-15), Intercellular Adhesion Molecule 1 (ICAM-1), and Angiopoietin-2(ANG2) in a sample of a subject, in combination with a)echocardiographic images of the heart obtained from said subject,wherein said images allow for assessing the presence or absence of (i)abnormal MFS, and/or (ii) LVH in said subject, or b) an echocardiographydevice which preferably allows for assessing the presence or absence of(i) abnormal MFS, and/or (ii) LVH in said subject, for predicting therisk of the subject of rapidly progressing to chronic heart failureand/or of hospitalization due to chronic heart failure and/or death.

The present invention also relates to the use of at least one bindingagent which binds to a biomarker selected from the group consisting of aBNP-type peptide, IGFBP7 (IGF binding protein 7), a cardiac Troponin,soluble ST2 (sST2), FGF-23 (Fibroblast Growth Factor 23), GrowthDifferentiation Factor 15 (GDF-15), Intercellular Adhesion Molecule 1(ICAM-1), and Angiopoietin-2 (ANG2) in a sample of a subject, incombination with a) echocardiographic images of the heart obtained fromsaid subject, wherein said images allow for assessing the presence orabsence of abnormal (i) abnormal MFS, and/or (ii) LVH in said subject,or b) an echocardiography device which preferably allows for assessingthe presence or absence of (i) abnormal MFS, and/or (ii) LVH in saidsubject, for predicting the risk of the subject of rapidly progressingto chronic heart failure and/or of hospitalization due to chronic heartfailure and/or death.

Preferably, the echocardiography device as referred to herein is usedfor the assessment of the presence or absence of (i) abnormal MFS,and/or (ii) LVH.

The term “binding agent” has been defined above. Preferably, the bindingagent binds specifically to a biomarker as referred to herein. Morepreferably, the binding agent is an antibody or a fragment thereof. Atleast one binding agent may be used. If the level of more than onebiomarker shall be determined, more than one binding agent is used. E.g.if the levels of a BNP-type peptide and IGFBP7 shall be measured, abinding agent which binds to the BNP-type peptide and an binding agentwhich binds to IGFBP7 is used.

Furthermore, a device adapted for carrying out the method of the presentinvention is provided, said device comprising

-   -   a) an analyzer unit comprising at least one binding agent for        measuring the level of at least one marker selected from the        group consisting of a BNP-type peptide, IGFBP7 (IGF binding        protein 7), a cardiac Troponin, soluble ST2 (sST2), FGF-23        (Fibroblast Growth Factor 23), Growth Differentiation Factor 15        (GDF-15), Intercellular Adhesion Molecule 1 (ICAM-1), and        Angiopoietin-2 (ANG2) in a sample of a subject,    -   b) a data input means for inputting the information whether the        subject suffers from (i) abnormal MFS, and/or (ii) LVH, and    -   c) an evaluation unit for comparing the measured level(s) with        reference level(s), whereby based on the results of comparing        the measured level(s) with the reference level(s), and based on        the information whether the subject suffers from (i) abnormal        MFS, and/or (ii) LVH, the risk of the subject of rapidly        progressing to chronic heart failure and/or of hospitalization        due to chronic heart failure and/or death is predicted, said        unit comprising a database with a reference level (or levels)        and a computer-implemented algorithm carrying out the        comparison.

The term “device” as used herein relates to a system comprising theaforementioned units operatively linked to each other as to allow thediagnosis according to the methods of the invention. Preferred bindingagents which can be used for the analyzer unit are disclosed elsewhereherein. The analyzer unit, preferably, comprises said agents inimmobilized form on a solid support which is to be contacted to thesample comprising the biomarkers the level of which is to be measured.Moreover, the analyzer unit can also comprise a detector which measuresthe level of detection agent which is specifically bound to thebiomarker(s). The measured level(s) can be transmitted to the evaluationunit.

According to some embodiments, an analyzer unit may be configured foroptical detection of an analyte, for example a marker, with a sample. Anexemplary analyzer unit configured for optical detection comprises adevice configured for converting electro-magnetic energy into anelectrical signal, which includes both single and multi-element or arrayoptical detectors. According to the present disclosure, an opticaldetector is capable of monitoring an optical electro-magnetic signal andproviding an electrical outlet signal or response signal relative to abaseline signal indicative of the presence and/or concentration of ananalyte in a sample being located in an optical path.

Said evaluation unit preferably, comprises a data processing element,such as a computer or computing device. Preferably, said element has animplemented algorithm for carrying out a comparison of said level(s) toa reference level (or reference level), wherein based on the results ofthe comparison and based an the information whether the subject suffersfrom abnormal midwall fractional shortening, the risk of a subject ofrapidly progressing to chronic heart failure and/or of hospitalizationdue to chronic heart failure and/or death is predicted. The results maybe given as output of parametric diagnostic raw data. It is to beunderstood that these data will usually need interpretation by theclinician. However, also envisaged are expert system devices wherein theoutput comprises processed diagnostic raw data the interpretation ofwhich does not require a specialized clinician.

It follows from the above that according to some embodiments of theinstant disclosure, portions of some steps of methods disclosed anddescribed herein may be performed by a computing device. A computingdevice may be a general purpose computer or a portable computing device,for example. It should also be understood that multiple computingdevices may be used together, such as over a network or other methods oftransferring data, for performing one or more steps of the methodsdisclosed herein. A computing device has access to a memory. A memory isa computer readable medium and may comprise a single storage device ormultiple storage devices, located either locally with the computingdevice or accessible to the computing device across a network, forexample. The computing device may also have access to an output device.Exemplary output devices include fax machines, displays, printers, andfiles, for example. According to some embodiments of the presentdisclosure, a computing device may perform one or more steps of a methoddisclosed herein, and thereafter provide an output, via an outputdevice, relating to a result, indication, ratio or other factor of themethod.

According to embodiments of the instant disclosure, software may includeinstructions which, when executed by a processor of the computingdevice, may perform one or more steps of the methods disclosed herein.Some of the instructions may be adapted to produce signals that controloperation of other machines and thus may operate through those controlsignals to transform materials far removed from the computer itself.

The definitions given herein below apply mutatis mutandis to thefollowing embodiments of the present invention.

In addition, the present invention relates to a method for predictingthe risk of a subject of rapidly progressing to chronic heart failureand/or of hospitalization due to chronic heart failure and/or death,said method comprising:

-   -   (a) measuring the level of at least one biomarker selected from        the group consisting of a BNP-type peptide, IGFBP7 (IGF binding        protein 7), a cardiac Troponin, soluble ST2 (sST2), FGF-23        (Fibroblast Growth Factor 23), Growth Differentiation Factor 15        (GDF-15), Intercellular Adhesion Molecule 1 (ICAM-1), and        Angiopoietin-2 (ANG2) in a sample from a subject who suffers        from (i) abnormal MFS, and/or (ii) LVH, and    -   (b) comparing the level of said at least one biomarker to a        reference level.

Preferably, the risk is predicted by carrying out the further step c) ofpredicting the risk of the subject of rapidly progressing to chronicheart failure and/or of hospitalization due to chronic heart failureand/or of death. Said step is based on the results of step b).

The subject who suffers from abnormal MFS (alternative (i)) may or maynot suffer from LVH. In particular, it is envisaged that the subjectdoes not suffer from LVH.

Preferred biomarkers and biomarker combinations for alternatives (i) and(ii) are disclosed elsewhere herein. Moreover, the reference level aswell as preferred diagnostic algorithms are described elsewhere herein.Preferably, a level (or levels) of the at least one biomarker above thereference level(s) in indicates that the subject is at risk of rapidlyprogressing to chronic heart failure and/or of hospitalization due tochronic heart failure and/or death. Also preferably, a level (or levels)of the at least one biomarker below the reference level(s) indicatesthat the subject is not at risk of rapidly progressing to chronic heartfailure and/or of hospitalization due to chronic heart failure and/ordeath.

The present invention also relates to the use at least one biomarkerselected from the group consisting of a BNP-type peptide, IGFBP7 (IGFbinding protein 7), a cardiac Troponin, soluble ST2 (sST2), FGF-23(Fibroblast Growth Factor 23), Growth Differentiation Factor 15(GDF-15), Intercellular Adhesion Molecule 1 (ICAM-1), and Angiopoietin-2(ANG2) in a sample of a subject who suffers from (i) abnormal MFS,and/or (ii) LVH for predicting the risk of the subject of rapidlyprogressing to chronic heart failure and/or of hospitalization due tochronic heart failure and/or death.

The present invention also relates to the use at least one binding agentwhich binds to a biomarker selected from the group consisting of aBNP-type peptide, IGFBP7 (IGF binding protein 7), a cardiac Troponin,soluble ST2 (sST2), FGF-23 (Fibroblast Growth Factor 23), GrowthDifferentiation Factor 15 (GDF-15), Intercellular Adhesion Molecule 1(ICAM-1), and Angiopoietin-2 (ANG2) in a sample of a subject who suffersfrom (i) abnormal MFS, and/or (ii) LVH for predicting the risk of thesubject of rapidly progressing to chronic heart failure and/or ofhospitalization due to chronic heart failure and/or death.

All references referred to above are herewith incorporated by referencewith respect to their entire disclosure content as well as theirspecific disclosure content explicitly referred to in the abovedescription.

EXAMPLES

The following Examples shall illustrate the invention. They shall,however, not be construed as limiting the scope of the invention.

Example 1

The combination of several circulating biomarkers with abnormal midwallfractional shortening (MFS), an early echocardiographic indicator ofpreclinical systolic dysfunction, with adverse outcome was investigated.

Circulating levels of various biomarkers (NTproBNP, ICAM1, cTNT, FGF23,IGFBP7, MMP2, PLGF, eSelectin, GDF15, ST2, Galectin-3,Vitamin D, CRP,CystatineC, OPN, P1NP, Mimecan, Endostatin, proANP, ANG2) were measuredin 550 elderly individuals (age 65-84 years) selected from the inPREDICTOR study. Participants were referred to cardiology centers forclinical examination and comprehensive Doppler echocardiography withcentrally-measured MFS. Absolute numbers of all cause death wereavailable after a median follow-up of 46 [39-54] months fromrecord-linkage of administrative data. Death was recorded in 36 cases.

Individuals with LV midwall dysfunction (MFS<15%) had higher levels ofNT-proBNP, cTnT, IGFBP7, sST2, GDF15, ICAM1 or FGF23 than those withnormal function. The relative incidence of mortality among individualswith abnormal MFS and elevated NT-proBNP (>75th age- and sex-specificpercentile) was 14.29% vs. 1.96% (abnormal MFS, low NT-proBNP), 6.22%(normal MFS, high NT-proBNP) and 0.85% (normal MFS, low NT-proBNP) (FIG.1).

Corresponding values for cTnT (>3 ng/L) were 14.09, 0.0, 4.65 and 3%(FIG. 2). Corresponding values for IGFBP7 above median concentrationwere 15.85, 2.94, 6.87 and 1.36% (FIG. 3). Corresponding values for sST2above median concentration were 15.89 4.82, 6.13 and 2.03% (FIG. 4).Corresponding values for FGF23 above median concentration were 14.04,6.58, 6.9 and 1.83% (FIG. 5).

After adjustment for sex, age, eGFR and history of hypertension,subjects with abnormal MFS and elevated NTproBNP, IGFBP7, FGF23, GDF15,ICAM1, ANG2 or sST2 had a higher risk of mortality compared to thosewith normal MFS and low NT proBNP, IGFBP7, FGF23, GDF15, ICAM1 , ANG2 orsST2.

Hazard Ratio (HR)=11.4 for NTproBNP (p=0.002), HR=6.1 for ST2 (p=0.005),HR=7.4 (p=0.002) for ICAM1, HR=3.2 (p=0.02) for Angiopoietin2, HR=6.5(p=0.002) for IGFBP7, HR=7.46 (p=0.002) for FGF23, HR=7.39 (p=0.002) forGDF15 (Table 1).

Elevation in of circulating NTproBNP, cTnT, IGFBP7, FGF23, GDF15, ICAM1,ANG2 or sST2 combined with early alterations in LV systolic functionwith MFS identify a subgroup of individuals in the general populationaged 65 or more at higher risk for death.

After adjustment for sex, age, eGFR and history of hypertension,subjects with abnormal MFS and elevated NTproBNP in combination withelevated ST2 or IGFBP7 or GDF15 or FGF23 had a higher risk of mortalitycompared with those with normal MFS and low NTproBNP, ST2, FGF23, GDF15,IGFBP7. HR=20.01 for NTproBNP and IGFBP7, HR=19.07 for NTproBNP and ST2,10.54 for NTproBNP and FGF23, HR=12.62 for NTproBNP and GDF15 (Table 1).After adjustment for sex, age, eGFR and history of hypertension,subjects with abnormal MFS and elevated IGFBP7 in combination withelevated ST2 had a higher risk of mortality compared with those withnormal MFS and low IGFBP7 and low ST2. HR=17.96 for IGFBP7 and ST2(Table 1).

Elevated circulating NTproBNP showed the most pronounce association toadverse outcome when measured combined with abnormal MFS (Table 1).Elevated circulating NTproBNP showed the most pronounced association toadverse outcome/mortality when measured in combination with elevatedcirculating IGFBP7 and either abnormal MFS or LVH (FIGS. 11 and 12,Table 1).

After adjustment for sex age eGFR and history of hypertension insubjects with abnormal MFS and elevated NTproBNP, Ang2, ICAM1, IGFBP7,sST2, GDF15 and FGF23 were found to have a significant higher risk formortality (Table 1).

In contrast several other biomarkers included in the presentinvestigation were not found to be suited when measured combined withabnormal MFS to identify a subgroup in the elderly population at higherrisk for CV/HF related adverse outcome.

Elevated CRP, CysC, Vitamin D and proANP were not found to be pronouncedassociated with higher risk of death in subjects with abnormal MFS.After adjustment for sex, age, egFR and history of hypertension insubjects with abnormal MFS and elevated CRP, CysC, proANP Vitamin D werenot found to have significant higher risk for mortality compared tothose with no abnormal MFS and low respective biomarker levels (Table1).

Example 2

The combination of the biomarkers referred to in Example 1 with abnormalMFS was investigated further in a subset of samples derived fromparticipants of the PREDICTOR study with normal left ventricular mass.

Individuals with LV midwall dysfunction (MFS<15%) had higher levels ofNT-proBNP, cTnT, IGFBP7, sST2 or FGF23 than those with normal function.The relative incidence of mortality among individuals with abnormal MFSand elevated NT-proBNP (>75th age- and sex-specific percentile) was13.51% vs. 0% (abnormal MFS, low NT-proBNP), 6.77% (normal MFS, highNT-proBNP) and 1.06% (normal MFS, low NT-proBNP) (FIG. 6). Correspondingvalues for IGFBP7 above median concentration were 16.0%, 6.93% and 1.9%(FIG. 8).

Absolute numbers of death among individuals with abnormal MFS andelevated cTnT (>3 ng/L) were 5 vs. 0 (abnormal MFS, low cTnT), 8 (normalMFS, high cTnT) and 2 (normal MFS, low cTnT)(FIG. 7). Correspondingnumbers for sST2 above median concentration were 3,2, 9 and 1.Corresponding numbers for FGF23 above median concentration were 5, 0, 7and 3. (FIG. 9) (FIG. 10).

Elevation in of circulating NTproBNP, cTnT, IGFBP7, FGF23 or sST2combined with early alterations in LV systolic function with MFSidentify a subgroup of individuals in the general population aged 65 ormore with normal left ventricular mass at higher risk for CV/HF relatedhospitalization/death and/or all cause death.

In contrast several other biomarkers included in the presentinvestigation were not found to be suited when measured combined withabnormal MFS in subjects with normal LV mass to identify a subgroup inthe elderly population at higher risk for CV/HF related adverse outcome.

Example 3

The combination of several circulating biomarkers with left ventricularhypertrophy, an early echocardiographic indicator of preclinicalstructural heart disease, with adverse outcome was investigated.

Circulating levels of NT-proBNP, ICAM-1, ST2, hsCRP, CystatinC, proANP,Vitamin D, FGF23, GDF15 and IGFBP7 were measured in 550 elderlyindividuals (age 65-84 years) selected from the in PREDICTOR study.Absolute numbers of death were available after a median follow-up of 46[39-54] months from record-linkage of administrative data. death wasrecorded in 36 cases.

Individuals with LVH had higher levels of NT-proBNP and IGFBP7, or thanthose with normal function. The relative incidence of mortality amongindividuals with LVH, elevated NTproBNP (>75^(th) age and sex specificpercentiles) and elevated IGFBP7 (above median concentration) was 19.15%vs. 0% (LVH, low NTproBNP, low IGFBP7), 13.79% (no LVH, high NTproBNP,high IGFBP7) and 1.08% (no LVH, low NTproBNP, lowIGFBP7) (FIG. 11). Therelative incidence of mortality among individuals with abnormal MFS,elevated NTproBNP (>75^(th) age and sex specific percentiles) andelevated IGFBP7 (above median concentration) was 17% vs. 3% (no abnormalMFS, low NTproBNP, low IGFBP7).

Elevation in of circulating NTproBNP and IGFBP7 combined with LVHidentify a subgroup of individuals in the general population aged 65 ormore at higher risk for death.

Elevation of both circulating NTproBNP and IGFBP7 combined with LVHidentify a subgroup of individuals in the general population aged 65 ormore at higher risk for death.

After adjustment for sex, age, eGFR and history of hypertension,subjects with LVH and elevated NTproBNP and IGFBP7, had a higher risk ofCV/HF related hospitalization/mortality compared to those with no LVHand low IGFBP7, NT proBNP.

Elevation of circulating NTproBNP, IGFBP-7, ICAM-1, FGF23, GDF15 or sST2combined with LVH identify a subgroup of individuals in the generalpopulation aged 65 or more at higher risk for CV/HF relatedhospitalization/death and/or all cause death.

After adjustment for sex, age, eGFR and history of hypertension,subjects with LVH and elevated FGF23, GDF15, ICAM1, or sST2 had a higherrisk of mortality compared to those with normal LV mass and low FGF23,GDF15, ICAM1 or sST2.

Hazard Ratio (HR)=3.5 for ST2 (p=0.08), HR=5.5 (p=0.009) for ICAM1,HR=4.9 (p=0.01) for FGF23, HR=5.83 (p=0.03) for GDF15 (Table 2).

After adjustment for sex, age, eGFR and history of hypertension,subjects with LVH and elevated NTproBNP in combination with elevated ST2or IGFBP7 or GDF15 or FGF23 had a higher risk of mortality compared withthose with normal LV mass and low NTproBNP, ST2, FGF23, GDF15, IGFBP7.HR=9.8 for NTproBNP and IGFBP7, HR=11.9 for NTproBNP and ST2, HR=8.3 forNTproBNP and FGF23, HR=7.1 for NTproBNP and GDF15 (Table 2). Afteradjustment for sex, age, eGFR and history of hypertension, subjects withLVH and elevated IGFBP7 in combination with elevated ST2 had a higherrisk of mortality compared with those with normal MFS and low IGFBP7 andlow ST2. HR=5.7 for IGFBP7 and ST2 (Table 2).

In contrast several other biomarkers included in the presentinvestigation were not found to be suited when measured in subjects withLVH to identify a subgroup in the elderly population at higher risk forCV/HF related adverse outcome.

Elevated CRP, CysC, Vitamin D and proANP were not found to be pronouncedassociated with higher risk of death in subjects with LVH. Afteradjustment for sex, age, egFR and history of hypertension in subjectswith LVH and elevated CRP, CysC, proANP Vitamin D were not found to havesignificant higher risk for mortality compared to those with no LVH andlow respective biomarker levels (Table 2).

TABLE 1 Multivariable Cox models for mortality in subjects with abnormalMFS and elevated concentrations of biomarkers Abnormal MFS P covariates:and elevated HR sex, age, eGFR and biomarker [95% CI] history ofhypertension ICAM-1 7.4 0.002 ST2 6.1 0.005 NT-proBNP 11.4 0.02 ANG-23.2 0.02 IGFBP7 6.5 0.02 hsCRP 1.1 0.84 Cystatin C 1.54 0.37 proANP 1.150.75 Vitamin D 2.2 0.13 FGF23 7.46 0.0017 GDF15 7.39 0.002 NTproBNP +IGFBP7 20.01 0.003 NTproBNP + ST2 19.07 0.005 NTproBNP + FGF23 10.540.025 NTproBNP + GDF15 12.62 0.016 IGFBP7 + ST2 17.96 0.006

TABLE 2 Multivariable Cox models for mortality in subjects with LVH andelevated concentrations of biomarkers P covariates: LVH and HR sex, age,eGFR and elevated biomarker [95% CI] history of hypertension ICAM-1 5.50.009 ST2 3.53 0.08 NT-proBNP 7.6 0.05 hsCRP 1.0 0.9 Cystatin C 0.980.97 proANP 0.7 0.5 Vitamin D 1.1 0.8 FGF23 4.9 0.01 GDF15 5.83 0.03NTproBNP + IGFBP7 9.8 0.03 NTproBNP + ST2 11.9 0.02 NTproBNP + FGF23 8.30.04 NTproBNP + GDF15 7.1 0.06 IGFBP7 + ST2 5.7 0.04

TABLE 3 Reference levels used in all investigations relating to elevatedbiomarkers in combination with LVH or abnormal MFS and a death BiomarkerElevated concentration NT-proBNP (pg/mL) >75th age- and sex-specificpercentiles, <40Years 40-50 Years >50 Years Female 63.6 63.4 73.2 Male25.2 29.9 45.7 hsCRP (mg/L) > median concentration (1,63) CysC (mg/L) >median concentration (1,09) VitaminD (ng/mL) > median concentration(9,57) IGFBP7 (ng/mL) > median concentration (171,9) sST2 (ng/mL) >median concentration (15) GDF15 (ng/L) > median concentration (1646)FGF23 (RU/mL) > median concentration (76,27) hs-cTnT (pg/mL) > 3 ICAM1 >median concentration (218) (ng/mL) ANG2 > median concentration (1,93)(ng/mL)

1-14. (canceled)
 15. A method for predicting the risk of a subject ofrapidly progressing to chronic heart failure and/or of hospitalizationdue to chronic heart failure and/or of death, said method comprising:(a) assessing in said subject (i) the presence or the absence ofabnormal midwall fractional shortening (abnormal MFS), and/or (ii) thepresence or the absence of Left ventricular hypertrophy (LVH) (b)measuring the level of at least one biomarker selected from the groupconsisting of a BNP-type peptide, IGFBP7 (IGF binding protein 7), acardiac Troponin, soluble ST2 (sST2), FGF-23 (Fibroblast Growth Factor23), Growth Differentiation Factor 15 (GDF-15), Intercellular AdhesionMolecule 1 (ICAM-1) and Angiopoietin-2 (ANG2) in a sample from thesubject, and (c) comparing the level of said at least one biomarker to areference level.
 16. The method of claim 15, wherein said subject doesnot show symptoms of heart failure.
 17. The method of claim 15, whereinsaid subject suffers from heart failure classified as stage A or stage Baccording to the ACC/AHA classification.
 18. The method of claim 15,wherein the presence or the absence of abnormal midwall fractionalshortening is assessed, and wherein said subject does not suffer fromleft ventricular hypertrophy.
 19. The method of claim 15, wherein saidsubject is 65 years or older.
 20. The method of claim 15, wherein theBNP-type peptide is BNP or NT-proBNP, and/or wherein the cardiacTroponin is Troponin T or Troponin I.
 21. The method of claim 15,wherein the subject is a human subject, and/or wherein the sample ablood, blood plasma or a blood serum sample.
 22. The method of claim 15,wherein the risk of a subject of progressing to chronic heart failureand/or of hospitalization due to chronic heart failure and/or of deathwithin a window period of 3 years is predicted.
 23. The method of claim15, wherein according to step a), alternative (i), an abnormal MFS ispresent in said subject, or wherein according to step (a), alternative(ii), LVH is present in said subject
 24. The method of claim 23, whereina level of the at least one biomarker above the reference level inindicates that the subject is at risk of rapidly progressing to chronicheart failure and/or of hospitalization due to chronic heart failureand/or of death, or wherein a level of the at least one biomarker belowthe reference level indicates that the subject is not at risk of rapidlyprogressing to chronic heart failure and/or of hospitalization due tochronic heart failure and/or of death.
 25. The method of claim 15,wherein (i) alternative (i) of step (a) is carried out, and wherein theat least one biomarker is selected from the group consisting of aBNP-type peptide, sST2, IGFBP7, a cardiac Troponin and FGF23 or (ii)alternative (ii) of step (a) is carried out, and wherein the at leastone biomarker is selected from the group consisting of IGFBP7 (IGFbinding protein 7), soluble ST2 (sST2), FGF-23 (Fibroblast Growth Factor23), and GDF15.
 26. A method for predicting the risk of a subject ofrapidly progressing to chronic heart failure and/or of hospitalizationdue to chronic heart failure and/or of death, said method comprising:(a) measuring the level of at least one biomarker selected from thegroup consisting of a BNP-type peptide, IGFBP7 (IGF binding protein 7),a cardiac Troponin, soluble ST2 (sST2), FGF-23 (Fibroblast Growth Factor23), Growth Differentiation Factor 15 (GDF-15), Intercellular AdhesionMolecule 1 (ICAM-1), and Angiopoietin-2 (ANG2) in a sample from asubject who suffers from (i) abnormal MFS, and/or (ii) LVH, and (b)comparing the level of said at least one biomarker to a reference level.27. The method of claim 26 wherein the levels of a BNP-type peptide andIGFBP7 are measured.
 28. The method of claim 26 wherein the presence orthe absence of Left ventricular hypertrophy (LVH) is assessed.
 29. Themethod of claim 26 wherein the presence or the absence of abnormal MFSis assessed.
 30. The method of claim 26 wherein the subject is 65 yearsor older.
 31. The method of claim 15 further comprising initiating atleast one suitable therapy if it is predicted that the subject is atrisk of rapidly progressing to chronic heart failure and/or ofhospitalization due to chronic heart failure and/or death.
 32. Themethod of claim 31 wherein the therapy is the administration ofangiotensin-converting enzyme (ACE) inhibitors and/or angiotensinreceptor antagonists (ARB) and/or aldosterone antagonists, and/ordiuretics and/or beta blockers.